Seborrheic dermatitis due to Malassezia species in Ahvaz, Iran.

BACKGROUND AND OBJECTIVE
Seborrheic dermatitis (SD) is a frequent disorder of the skin that is distinguished by the development of erythematous patches and yellow-gray scales. It is a multifactor disease that requires predisposing factors for its progress. Presence of these factors leads to reproduction of opportunistic yeast Malassezia spp. The aim of the present study was to isolate and identify distribution of Malassezia species on the scalp of SD patients in Ahvaz using modified Dixons agar.


MATERIALS AND METHODS
A total of 110 patients diagnosed with SD were sampled. The sampling was carried out by brushing the hair and collecting the dandruff in paper pockets. For identification of Malassezia species, the scalp scales were cultured in Dixons agar. A combination of different characteristics including yeast cell morphology, ability to grow on Sabouraud dextrose agar, catalase test and ability to utilize individual Tweens (20, 40, 60 & 80) were used for identification of species.


RESULTS
Twenty-seven of 110 (24.5%) SD patients had positive cultures for Malassezia species of which 17 (63%) were male and 10 (37%) were female. The most commonly identified Malassezia species was M. globosa (40.7%) followed by M. pachydermatis (22.2%), M. furfur (11.1%) and M. restricta(7.4%) and Malassezia species (18.5%).


CONCLUSION
Malassezia globosa was considered to be the most important orgaism involved in cases with Seborrheic dermatitisin this study.


INTRODUCTION
Superficial mycoses are fungal infections that invade keratinized layer of skin and hair shafts of the human body. These infections usually are asymptomatic and without cellular and/or humoral responses. Pityriasis versicolor is a chronic and mild superficial mycosis caused by several species of Malassezia, a human normal flora. Pityriasis versicolor has worldwide distribution; however it is more prevalent in tropical and subtropical areas. Several reports showed that its prevalence is 20%-50% in tropical areas (1-3). Disease is more common in young people with age range 20-30 year, however it is not uncommon in children in tropics (4). Pityriasis versicolor is one of the most common fungal infections in Iran, especially in north and south, south west of the country that have tropical conditions (5)(6)(7)(8). In addition, chronic infections, corticosteroid therapy, pregnancy and genetically factors have important role in disease.
Malassezia species are associated with pityriasis versicolor, folliculitis, Seborrheic dermatitis (SD), and atopic dermatitis (9, 10). In addition, Malassezia colonize healthy skin in human and some animals. Seborrheic dermatitis (SD) is a frequent disorder of the skin that is distinguished by the development of erythematous patches and yellow-gray scales. Seborrheic dermatitis is a multifactor disease that requires predisposing factors for its progress. Presence of these factors leads to reproduction of opportunistic yeast Malassezia spp.
Seborrheic dermatitis is usually occurring in young adults and disease is most often seen on the areas of the body rich in sebaceous glands (face, scalp, upper trunk) (11,12). Dandruff and SD are associated with the presence of sebum, Malassezia metabolism, and individual susceptibility (13). The distribution disease is overall 1-5% suggested and can affect any ethnicity (12). However disease is more prevalent in male than female.
Most of the Malassezia species are lipophilic organisms and are part of human normal flora, especially greasy (oily) skin. Currently more than 14 species of Malassezia detected as causative agents of pityriasis versicolor, however the most common agents are M. globosa, M. furfur, M. obtusa, and M. sympodialis (14-16). Other agents are as follows; M. restricta, M. slooffiae, M. pachydermatis, M. dermatis, M. japonica, M. nana, M. yamatoensis, M. equina, M. caprae, and M. cuniculi (4, 11, 16). The aim of the present study was to isolate and identify Malassezia species from SD using modified Dixons agar in the university students of medical sciences in Ahvaz.
Sampling and culture. In the present study 110 medical students diagnosed with SD (not simple dandruff) (64 male and 46 female) were sampled. Age, gender and disease extent for each student were recorded. Scalp scales were collected in sterile sampling pockets using hairs brushing. Samples were transferred to the medical mycology laboratory, Ahvaz Jundishapur University of Medical Sciences. Scalp scales were cultured in two sets of Dixons agar tubes and incubated at 37°C for two weeks aerobically and considered every 2-3 days for growing.
Isolation and identification procedures. Cultured tubes were examined every two days for growth. The Malassezia isolates were detected by its morphological and biochemical and physiological characteristics. For each positive sample several diagnostic tests including grow on the medium without lipid supplementation (Sabouraud dextrose agar, SDA (Merck, Germany), lipid-dependent species by the Tween assimilation method (Tween 20, 40, 60 and 80) and catalase reaction (2, 10, 17).

Production of brown colony on Dixon agar. Deep brown pigmentation by M. furfur was observed on
Dixons agar with tryptophan, whereas other species produced no pigmentation in medium (17).
Cell morphology. A direct smear from grown colonies in Dixon agar after 10-14 days at 37°C was prepared. Yeast cell morphology was studied by methylene blue stained smears.
Growth on SDA. Sabouraud dextrose agar slants inoculated with a portion of colony of Malassezia grown on Dixons agar and incubated at 37°C for two weeks. Grow on SDA medium was assessed by the presence of white-cream and brittle colonies of Malassezia.
Catalase reaction. A drop of hydrogen peroxide (H 2 O 2 ) was put on a portion of a colony on a clean glass slide and the production of gas bubbles indicated a positive reaction for catalase.

Ability to utilize Tween. A suspension of each
Malassezia species in sterile distilled water was prepared and adjusted to about 10 5 cell/ml. Two milliliter of suspension was added into 16 ml melted sterile SDA and vigorously mixed and poured into a 9 cm diameter petri dish. Plates were put at ambient temperature for solidification. Then four wells with 2 mm diameter punch in each plates and filled with 5 µl of sterile Tweens 20, 40, 60 and 80 respectively. Plates were incubated at 32°C for one week. The degree of growth (precipitation) around each well indicates utilization of Tween by Malassezia (2, 17).

DISCUSSION
Although the exceptional cause of SD is unknown, increase of Malassezia population in skin has been described as an important contributing factor (12). All Malassezia species (exception M. pachydermatis) are able to degrade lipids in sebum, consume certain saturated fatty acids and produce free fatty acids and triglycerides. Several studies have shown that SD is a chronic disease that affects adults, adolescents and infants (12,18,19). Many investigators and clinicians believe that Malassezia spp play an important role in the pathogenesis of SD (20, 21).
The incidence of SD in general population is approximately 11.6%, whereas this rate in infants in the first three months of life is 70% (22). In a study from Iran conducted by Zarrin et al. (8) SD was considered as the common superficial fungal disease among primary school students in Ahvaz. In our study only 24.5% of examined patients had positive culture for Malassezia species. It is indicated that another factors associated with SD in our patients and Malassezia species have few role in SD in this area. However, Lee et al. (23) report showed that Malassezia species play an important role in the pathogenesis of Korean SD patients and 85% of them contaminated to Malassezia species. In a study conducted by Hedayati et al. (24), 77% of the SD specimens were yielded Malassezia in north of Iran. The rate of recovery of Malassezia organisms from SD patients is higher in this study compare with our result, probably because of different climate in north of Iran which is very humid.
Del Rosso (12) in a report from the USA believes that most cases of SD appear in men than women and this rate was 1.7 in our study. In addition, the most cases of SD with positive culture were distributed at age range 21-25 years old. Several reports showed that the rate of Malassezia species in the twenties with SD is high because of sebaceous glands greatest activity (24,25). Seborrheic dermatitis is a chronic disease and some cases may be more persistent with recurrences over several months (12). Our study shows that 66.4% of patients had duration more than one year whereas 10.9% affected less than six months.
Different Malassezia species were reported as causative agents of SD in the different countries. Lee et al. (23) reported M. restrictaas the most important species in Korean SD patients. In addition, Prohic (26) in a study from Bosnia and Herzegovina believes that M. restricta (27.5%) is the main agents of SD and M. globosa (17.5%) and M. slooffiae (15%) are the next agents. In a molecular study by Tajima et al. (11), M. restricta and M. globosa were detected as the predominate agents of SD. In contrast, in Hedayati et al. study in north of Iran M. globosa was reported as the most frequently agent on scalp and face lesions, whereas M. furfur had most frequency on trunk lesions (24). In the present study, out of the 110 scalp scales that were cultured on Dixons agar, 24.5% yielded Malassezia that the most frequently Malassezia species was M. globosa (40.7%), followed by M. pachydermatis (22.2%), M. furfur (11.1%) and M. restricta (7.4%).
In conclusion, the results of present study showed low recovery rate (24.5%) of Malassezia species on patients with SD. In addition Malassezia globosa is considered to be the most important Malassezia species in our SD patients.